An Assembly-Line Mechanism for In Vitro Encapsulation of Fragmented Cargo in Virus-Like Particles

The ability of virus shells to encapsulate a wide range of functional cargoes, especially multiple cargoes─siRNAs, enzymes, and chromophores─has made them an essential tool in biotechnology for advancing drug delivery applications and developing innovative new materials. Since therapeutic cargo may be formulated in different physical states (size, surface charge, etc.) we have investigated the spontaneous encapsulation of multiple, charged, small nanoparticles which repel when free in solution, inside a spherical cage formed of brome mosaic virus (BMV) coat proteins. Unlike the cases of coassembly of virus-like particles (VLPs) from multiple oligonucleotides and single nanoparticle cargo, the structure of virus-like particles thus obtained is consistent with that of the native icosahedral BMV capsid. Working with small metal nanoparticles as cargo allowed the pathway of assembly to be followed by electron and liquid atomic force microscopy and cryoelectron tomography. Based on the structural identification of nanoparticle─BMV protein intermediates, we have found that multiple cargo encapsulation occurs in stages through a specific "assembly line" pathway that is different from the previously described in vitro assembly mechanisms of virus-like particles (VLP). We propose a model that explains the experimental findings, some of which will be important for delivery applications, for instance, the pronounced nanoparticle size selectivity in competition experiments where different nanoparticle sizes are present.